(1) Field of the Invention
This invention relates generally to recombinant polypeptides, and polynucleotides encoding those peptides, that arrest proliferating cells in mitosis, and to methods of treating patients with hyperplasias by administering pharmaceutical compositions comprising recombinant polypeptides or polynucleotides.
(2) Description of the Related Art
Cell proliferation in normal cells is regulated by growth factors that signal the cell to re-enter the cell cycle from a state of quiescence. For example, when wounding occurs, growth factors, e.g., platelet-derived growth factor, stimulate fibroblasts to proliferate. Under normal circumstances, withdrawal of growth factors causes proliferating cells to exit the cell cycle and become quiescent. Cancer cells (neoplasia) have lost the ability to exit the cell cycle and proliferate in the absence of growth factors. That is, the cancer cells think that they are receiving growth factor signals. On the other hand, most benign hyperplasias result from the hyperproliferation of cells due to the inappropriate or uncontrolled secretion of growth factors.
Transition across the G2-M border is crucial to advancement through the cell cycle. The G2-M transition is controlled by the activation of the cyclin-dependent kinase cdc2 by the cyclin B. For a review on the regulation of the cell cycle by the cyclin B/cdc2 complex, see Bastians et al., 1999). Basically, levels of cyclin B accumulate during interphase until they reach an optimal level near the end of G2, at which time, cyclin B binds to and activates the kinase cdc2. The active cyclin B/cdc2 complex phosphorylates various molecules that facilitate the entry into mitosis. The level of cyclin B/cdc2 within the nuclei of proliferating cells reach a maximum at metaphase, upon which time cdc2 activates the destruction of cyclin B. Upon the destruction of cyclin B, the cells are then able to undergo anaphase and hence complete the cell cycle.
Diseases of uncontrolled cell proliferation, or hyperplasias, are common health problems today. Examples of diseases of cell over-proliferation include psoriasis, seborrhea, eczema, benign prostate hyperplasia, congenital adrenal hyperplasia, endometrial hyperplasia, squamous cell (vulvular) hyperplasia, sebaceous hyperplasia, Crohn's Disease, leukemia, carcinoma, sarcoma, glioma, and lymphoma. Current treatment protocols for these diseases are generally toxic and/or ineffective as permanent cures.
Current cancer therapies involve the use of mitotic poisons, such as taxol or colcemid, or radiation to induce cell death in rapidly dividing cells. Such an approach attempts to kill cancer cells at a faster rate than healthy, naturally proliferating cells. Current forms of chemotherapy have severe side affects due to the destruction of healthy tissue, and are therefore a compromise at best.
Current treatments for other hyperplasias, such as benign enlarged prostate or psoriasis and eczema, are largely based upon the use of steroids or retinoids. For example, psoriasis is commonly treated by oral administration of drugs that engage receptors for glucocorticoids, retinoids and vitamin D, i.e., lipid soluble hormones which bind to nuclear receptors. Much of the current research in psoriasis treatment is now focused on the thiazolidinedione class of drugs that bind to the newly discovered peroxisome proliferator-activated receptor gamma. These drugs are administered orally and target multiple cellular processes, therefore leading to potential side effects. For a review on oral steroid-like treatments for psoriasis, see Pershadsingh, HA, 1999 and Ellis, et al., 2000.
Routine topical treatments for psoriasis and/ or eczema include steroid creams that are applied to the skin. However, the use of steroids is associated with several side effects including skin thinning, stretch marks and discoloration. Recently, a new cream called PROTOPIC®, which comprises an immunomodulatory drug called tacrolimus (Asakura, et al., 1999, U.S. Pat. No. 5,955,469), has been approved as a topical therapy for eczema. Although the use of a topical immunomodulator may have advantages over the use of steroidal based creams, immunomodulators exert their effects on the immune system. For individuals who are immunocompromised, the use of steroidal creams or creams containing immunosuppressing immunomodulators could possibly have deleterious effects.
Phototherapy, in the form of UVA or UVB irradiation, either used alone, or in combination with other forms of therapy, is a common treatment for inflammatory skin diseases. An important drawback to using ultraviolet light is the heightened risk of skin cancer in the recipient of such treatment. For a review of recent advances in phototherapy, see Simon, et al., 2000.
Many dermatological diseases such as, for example, psoriasis, eczema and seborrhoea, have two major components, inflammation and hyperproliferation. The current standard treatments for these diseases primarily target the inflammation aspect of the diseases.
There have been some efforts in the development of treatments targeting hyperproliferation of cells. Sato et al, have recently reported that the overexpression of platelet-activating factor receptor (PAFR) in transgenic mice leads to epidermal hyperproliferation that resembles psoriasis. Topical application of a cream comprising the PAFR antagonist WEB2086 to the transgenic mice resulted in the suppression of the number of proliferating cells (Sato, et al., 1999)
Benign prostate hyperplasia (BPH), or enlarged prostate, is another economically important disease of cell hyperproliferation. Over 30% of men in their 70s suffer from BPH and, while the growth in and of itself is harmless, it can lead to other serious urogenital disorders. For example, BPH causes problems in urination and can lead to serious kidney disorders, incontinence and impotence. Over 400,000 prostatectomies are performed each year in the United States. Even though surgical prostatectomy can cause bleeding, infection, impotence, retrograde ejaculation and incontinence, it still remains the predominant therapy for BPH.
In addition to the traditional surgical resection procedures, which are used to open the constricted urethral lumen or to remove the entire prostate, several new surgical methods are being developed for BPH. These novel surgical treatments include microwave thermotherapy, transurethral electrovaporization, laser ablation (Bolmsjo et al., 2000, Ohtani et al., 1999, Gilling et al., 1996). Although these treatments are effective toward correcting bladder retention symptoms, they are surgical and as such may increase the chances of secondary complications such as incontinence and impotence.
Other medical treatments for BPH include the administration of finastride (a 5-alpha reductase inhibitor) or alpha-adrenoceptor antagonists (alpha blockers). Finastride is a 4-aza steroid compound that inhibits the conversion of testosterone to dihydrotestosterone (Stoner, 1990). In clinical trials, finastride has been shown to increase urine flow rate by 30% and to decrease prostate size by 18%. However, users of finastride complain of impotence and decreasing libido, suggesting that this drug affects other physiological pathways (Carraro et al., 1996).
Other agents used to treat BPH are alpha blockers. Alpha-blockers are alpa-adrenergic receptor antagonists, which act by relaxing the smooth muscle cells of the prostate, thereby facilitating the flow of urine through the urethra. Additionally, other classes of alpha blockers have been shown to actually suppress prostate growth by inducing apoptosis of prostate epithelial cells. These novel pro-apoptotic effects of some alpha blockers are independent of the alpha-adrenoceptor antagonism (Kyprianou et al., 2000). Side effects of alpha blocker treatment include dizziness, headache, drowsiness and retrograde ejaculation.
The development of safe and effective drugs with specific cell proliferation inhibiting properties would bring significant improvements in the treatment of hyperplasia and cancer. Such drugs can be used alone or in conjunction with current conventional treatments for cancer or hyperplasias, to improve the safety and efficacy of those treatments. This invention is directed to polypeptide and polynucleotide based cell cycle effectors that specifically and narrowly block only the G2/M transition of actively proliferating cells, and the use thereof. By specifically affecting only those cells that have committed to undergo mitosis, the invention would be expected to have little to none side effects. Thus,the development and commercialization of new treatments that safely and effectively target the cellular hyperproliferation aspect of cancer and/or hyperplasia diseases is sorely needed.
(3) References and Related Art
(i) Patent Documents    Asakura et al., Sep. 21, 1999, U.S. Pat. No. 5,955,469. “Pharmaceutical composition.”    Beach et al., Dec. 9, 1997, U.S. Pat. No. 5,695,950. “Method of screening for antimitotic compounds using the cdc25 tyrosine phosphatase.”    Beach et al., Oct. 5, 1999, U.S. Pat. No. 5,962,316. “Cell-cycle regulatory proteins, and uses related thereto.”    Beach et al., Oct. 19, 1999, U.S. Pat. No. 5,968,821. “Cell-cycle regulatory proteins, and uses related thereto.”    Beach et al., Mar. 28, 2000, U.S. Pat. No. 6,043,030. “Cell-cycle regulatory proteins, and uses related thereto.”    Giordano, Dec. 19, 2000, U.S. Pat. No. 6,162,612. “Human cyclin-dependent kinase-like proteins and methods of using the same.”    Henderson et al., Dec. 16, 1997, U.S. Pat. No. 5,698,443. “Tissue specific viral vectors.”    Kauffman et al., May 24, 1994, U.S. Pat. No. 5,314,688. “Local delivery of dipyridamole for the treatment of proliferative diseases    Wu et al., Jun. 3, 1997, U.S. Pat. No. 5,635,383. “Method for the introduction of genes into mammalian cells by a soluble molecular complex comprising a receptor ligand and a polycation.”    Weiner et al., Jan. 9, 2001, U.S. Pat. No. 6,172,201. “Cellular receptor for HIV-1 Vpr essential for G2/M phase.”
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